Not applicable.
The field of this invention is the area of apoptosis (programmed cell death) and methods for the study of the regulation thereof. Specifically, the present invention provides an in vitro system for the analysis of apoptosis and specific regulators of the apoptotic pathway.
Apoptosis is a distinct form of cell death controlled by an internally encoded suicide program [reviewed by Steller, H. (1995) Science 267, 1445-1449; White, E. (1996) Gene and Dev. 10, 1-15]. Morphologic changes associated with apoptosis include condensation of nucleoplasm and cytoplasm, blebbing of cytoplasmic membranes, and fragmentation of the cell into apoptotic bodies that are rapidly phagocytosed by neighboring cells [Kerr, J. (1971) J. Pathol. 105, 13-20; Wyllie et al. (1980) Int. Rev. Cytol. 68, 251-305]. Biochemical markers of apoptosis include DNA fragmentation into nucleosomal fragments [Wyllie, A. (1980) Nature 284, 555-556], activation of the interleukin lb converting enzyme (ICE)-family of proteases [Duan et al. (1996) J. Biol. Chem. 271, 1621-1625; Wang et al. (1996) EMBO J. 15, 1012-1020], and cleavage of substrates of the ICE-family of proteases, including poly(ADP-ribose) polymerase (PARP) [Tewari et al. (1995) Cell 81, 801-809; Nicholson et al. (1995) Nature 376, 37-43] sterol regulatory element binding proteins (SREBPs) [Wang et al. (1995) J. Biol. Chem. 270, 18044-18050; Wang et al. 1996, supra], nuclear lamin [Lazebnik et al. (1995) Proc. Natl. Acad. Sci. USA 92, 9042-9046], and the U1 associated 70 kDa protein [Casciola-Rosen et al. (1994) J. Biol. Chem. 269, 30757-30760].
The cell suicide program is illustrated by genetic studies in the nematode Caenorhabditis elegans [Hengartner and Horvitz (1994) Philos. Trans. R. Soc. London Ser. B 345, 243-246]. Two genes involved in the control of programmed cell death in C. elegans have been well characterized. One gene (ced-9) encodes a protein that prevents cells from undergoing apoptosis [Hengartner et al. (1992) Nature 356, 494-499], and the ced-3 gene encodes a protease required for initiation of apoptosis [Yuan and Horvitz (1990) Dev. Biol. 138, 33-41].
The bcl-2 family of genes are mammalian counterparts of ced-9 [Hengartner and Horvitz (1994) Cell 76, 665-676]. Over-expression of bcl-2 coding sequences prevents mammalian cells from undergoing apoptosis in response to a variety of stimuli [reviewed by Reed, J. C. (1994) J. Cell Biol. 124, 1-6]. The BCL-2 protein is located primarily on the outer membranes of mitochondria [Monaghan et al. (1992) J. Hist. Cytochem. 40, 1819-1825; Krajewski et al. (1993) Cancer Res. 53, 4701-4714; de Jong et al. (1994) Cancer Res. 54, 256-260]. The presence of BCL-2 on the mitochondria surface is correlated with a block in the release of cytochrome c in response to triggers of apoptosis in cells which do not express the BCL-2 protein on the mitochondrial surface [Yang et al. (1997) Science 275, 1129-1132]. Holocytochrome c, but not apocytochrome c, triggers activation of CPP32 and the apoptotic cascade. Without wishing to be bound by theory, it is believed that the Bcl-2 protein inhibits apoptosis by preventing release of holocytochrome c from the mitochondrial membrane and also prevents depolarization of the mitochondrial membrane.
The CED-3 protein is a cysteine protease related to the ICE-family of proteases in mammalian cells [Yuan et al. (1993) Cell 75, 641-652]. The closest mammalian homolog of CED-3 is CPP32 [Fernandes-Alnemri et al. (1994) J. Biol. Chem. 269, 30761-30764], which cleaves PARP and SREBPs in cells undergoing apoptosis [Tewari et al. (1995) supra; Nicholson et al. (1995) supra; Wang et al. (1996) supra]. CPP32, which is also called caspase-3, is closely related to CED-3 in terms of amino acid sequence identity and substrate specificity [Xue and Horvitz (1995) Nature 377, 248-251]. Like CED-3 in C. elegans, CPP32 normally exists in the cytosolic fraction as an inactive precursor; that precursor is activated proteolytically in cells undergoing apoptosis [Schlegel et al. (1996) J. Biol. Chem. 271, 1841-1844, 1996; Wang et al. (1996) supra]. Further evidence for the requirement for active CPP32 in apoptosis is that a tetrapeptide aldehyde inhibitor that specifically inhibits CPP32 activity blocks the ability of cytosol from apoptotic cells to induce apoptosis-like changes in normal nuclei in vitro. [Nicholson et al. (1995) supra].
Triggering of apoptosis by activated CPP32 is part of the highly regulated mechanism for initiation of apoptosis; careful regulation of this pathway is necessary to prevent unwanted cell death. CPP32 is activated by multiple proteolytic cleavages of its 32 kDa precursor form, generating the 17/11 kDa or 20/11 kDa active form [Nicholson et al. (1995) supra; Wang et al. (1995) supra]. CPP32 is activated by cleavage at aspartic acid residues, a hallmark of ICE-like proteases [Thomberry et al. (1992) Nature 356, 768-774], and a cascade of ICE-like proteolytic cleavages leading to apoptosis has been proposed [Tewari et al. (1995) supra; Wang et al. (1996) supra]. Activated CPP32 from HeLa cell extracts cleaves the CPP32 precursor [Wang et al. (1996) supra], indicating that CPP32 can be activated through autocatalysis. Autocatalytic cleavage is probably responsible for active enzyme when the CPP32 precursor is expressed in large quantity in bacteria [Xue and Horvitz (1995) supra]. Recently, another ICE-family protease has been identified that may be responsible for cleaving the CPP32 precursor into the 20/11 kDa active form. This enzyme has been purified from hamster liver extracts and identified as the hamster homolog of Mch2a [Liu et al. (1996) J. Biol. Chem. 271, 13371-13376; Fernandes-Alnemri et al. (1995) Cancer Res. 55, 2737-2742]. Autocatalysis and the protease cascade may provide the signal amplification necessary for rapid and irreversible apoptosis, but the intracellular factors that trigger this amplification have yet to be identified.
There have been several previous reports of cell-free apoptosis systems that induce apoptotic changes in the added nuclei [Lazebnik et al. (1993) J. Cell Biol. 123, 7-22; Newmeyer et al. (1994) Cell 79, 353-364; Eeari et al. (1995) EMBO. J. 14, 5201-5208; Martin et al. (1995) EMBO J. 14, 5191-5200]. These systems require cytosol from cells that are already undergoing apoptosis in vivo; thus, they cannot be used to detect triggering factors.
There is a need in the art for in vitro methods for the analysis of compounds and biological factors which trigger or accelerate apoptosis or which interfere with the induction of apoptosis, as well as those which can increase the apoptotic effect of chemotherapeutic agents in cancers, especially those expressing oncogenic bcl-2. This need is met by the present invention, which allows the study of apoptosis and regulators thereof in a cell-free system in which the analysis is not complicated by previous induction of the apoptotic pathway in the cells used to prepare the test extracts.
The present invention provides an in vitro system and methods for the analysis of the regulation of apoptosis and for the identification of activators and inhibitors of the apoptotic pathway; the present system is improved over prior art systems for the study of apoptosis in that the prior art systems depended on cell free extracts prepared from organisms in which the apoptosis pathway had already been induced. Thus, the present system and methods permit freedom from the potential interference of apoptosis-inducing factors or other conditions on which prior art systems have relied.
As exemplified herein, the present invention provides an in vitro system for analysis of apoptosis and its regulation, where the test system includes a 100,000xc3x97g supernatant of HeLa cells from suspension culture (S-100). In its first aspect, the HeLa S-100, to which challenge compounds are added, is assayed for CPP32 proteolytic activity using radiolabeled poly(adenosine diphosphate-ribose polymerase (PARP) and radiolabeled sterol regulatory binding protein 2 (SREBP-2) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. The radiolabeled PARP and SREBP-2 can be prepared by in vitro translation in the presence of 35S-methionine as described in Example 3 herein. In a second aspect, the HeLa S-100, to which challenge compounds are added, is assayed for DNA fragmentation activity, by incubating the treated S-100 with hamster liver cell nuclei and then extracting the genomic DNA and analyzing by agarose gel electrophoresis. The specific proteolytic activity is accelerated by the addition of dATP or dADP (at a concentration from about 0.1 to about 2 mM, preferably about 1 mM.) DNA fragmenting activity is similarly dependent on the presence of dATP. It has been demonstrated that cytochrome c is required in the cell-free extract for the dATP-dependent activation of the apoptotic pathway, especially for the activation of the apoptosis marker protease.
The present invention provides a cell-free system which duplicates the features of the apoptotic program, including the activation of CPP32 and DNA fragmentation. Apoptosis in this system is initiated by the presence of soluble cytochrome c and dATP at sufficient concentrations. This system allows the fractionation and purification of the biochemical components that trigger the activation of the apoptotic proteases and DNA fragmentation.
The present invention further provides a method for identifying antagonists of dATP in the cytosol of adenosine deaminase-deficient cells, such as T cells from persons with severe combined immunodeficiency. dATP levels in adenosine deaminase-deficient cells are elevated in comparison to those of normal cells, and without wishing to be bound by any particularly, this is believed to contribute to the symptoms of the deficiency. Identification of antagonists of the initiation of apoptosis can lead to treatments for the amelioration of the clinical state of deficient individuals.
The present invention also provides methods for identification of compounds which trigger apoptosis even where the bcl-2 oncogene protein is present. The bcl-2 oncogene is associated with resistance to chemotherapy in human cancer, and compounds which cause CPP32 protease and DNA fragmentation nuclease-activation in bcl-2 oncogene extracts can be identified in the cell free assays of the present invention where the S-100 extract is prepared from BCL-2 expressing cells.
Additionally, the present invention allows the identification of compounds which effectively increase the apoptotic response to dATP and/or cytochrome c, including those which increase dATP levels in treated cells and -those which promote release of cytochrome c from mitochondrial membranes. Such compounds can be used to increase the effectiveness of chemotherapeutic agents which act by inducing apoptosis.